This research will be performed primarily in Buenos Aires, Argentina at The Institute of Experimental Medicine and Biology in collaboration with Dr. Marta Tesone as an extension of the P.l's NIH parent grant # RO1 HD20869-17. The goal of this project is to elucidate the REGULATION and ROLE OF APOPTOSIS IN LUTEAL REGRESSION during the ovarian cycle and pregnancy. Preliminary experiments have determined the dynamics of pro-apoptotic gene (e.g., caspases) expression and activation in the macaque corpus luteum (CL), pursuant to their regulation and role in controlling the functional lifespan of the CL. In the present project it is proposed to pursue further this novel research on apoptosis in the corpus luteum using a more general laboratory animal model, the rat. Accordingly, the specific aims of this project are: 1) To determine the changes in the pro-apoptotic genes (caspases -2, -3, -8 and -9) during the lifespan of rat corpus luteum in the estrous cycle and pregnancy, 2) To evaluate the regulation of these pro-apoptotic genes by the luteolytic hormone prostaglandin F2 alpha and 3) To determine whether the anti-apoptotic agent sphingosine-1-phosphate (S1P) will prevent caspase activation and luteolysis of the corpus luteum. The results of the proposed studies will facilitate the design of critical comparative studies to determine the relevance of these apoptotic pathways to the primate CL, and ultimately, clinical syndromes of luteal dysfuntion. To achieve these goals, the research design includes the study of caspases in CL obtained in different stages from cycling and pregnant rats. In addition, caspases will be studied in CL from pregnant rats treated with prostaglandin F2alpha (a luteolytic hormone) and/or sphingosine-1-phosphate (S1P: an antiapoptotic agent). The expression of caspase mRNA in CL will be analyzed by RT- Real Time PCR; and the caspase protein levels, activity and localization in the CL will be studied by Western blots, enzyme assays and ICC. Apoptosis will be measured by TUNEL and electrophoresis in agarose gels. [unreadable] [unreadable] [unreadable]